1. Field of the Invention
The present invention relates to a separation unit, and particularly to an apparatus for separating a specific component in a fluid. More particularly, the invention relates to an apparatus for separating a specific component, more specifically a lipid component contained in a high density lipoprotein (abbreviated as HDL hereinafter) fraction in a biological fluid such as blood. Furthermore, the present invention relates to a method and an apparatus for separating and measuring high density lipoproteins, which is used for measurement of such a component.
2. Background of the Invention
A method of separating a high density lipoprotein is disclosed in, for instance, JP-A-3-99268.
Because the HDL cholesterol in blood is a factor for coronary atherosclerosis prevention, measurement of the HDL cholesterol is performed as part of medical check for lifestyle-related diseases prevention in many facilities.
A total cholesterol in blood, plasma, and serum is one of well-known parameters used to estimate the risk relating to a degree of coronary artery diseases.
However, the concentration of the total cholesterols is the only defined value to estimate the risk of individual developing diseases.
It is more significant to measure both an amount of low density lipoproteins and an amount of high density proteins.
In the epidemiological and clinical studies, it has been clarified that there is a positive relationship between the LDL cholesterol and the coronary artery diseases, and also that there is a negative relationship between the HDL cholesterol and the coronary artery disease.
Because the relationship between the total cholesterol and the coronary artery disease is analogous to that between the HDL and the coronary artery disease, measurement of the HDL and total cholesterol is required for estimation of the risk of coronary artery diseases.
Such measurement as described above is advantageously employed in a wide range for diagnosis of coronary artery diseases.
HDL cholesterol is currently checked by fractionating HDL from other lipoproteins and measuring an amount of HDL cholesterol in the supernatant liquid by the enzyme method or the like.
To measure HDL cholesterol independently, it is necessary to separate other lipoproteins (LDL, VLDL (very low density lipoprotein), chylomicron) present in a biological sample. Embodiments of separation of lipoproteins other than HDL include a method making use of different surface charges (electrophoresis by using paper or agarose as a carrier) or a method making use of differences in apoliproteins (epidemiological method of using a specific antibody).
Furthermore, there are a method making use of a ultracentrifugal force, and a method of precipitating and removing lipoproteins other than HDL by using bivalent metal ions and polyanion.
Because it is necessary to use an ultracentrifugal separator in the ultracentrifugal force method, operation on the ultracentrifugal separator is complicated and it takes a long period of time. Therefore, a method of fractionating HDL used for measurement is mainly a precipitation method.
As a polyanion precipitant based on a combination of a bivalent metal ion and a polyanion, such combinations as heparin-Ca2+, heparin-Mn2+, phosphotungustic acid-Mg2+, and dextran sulfate-Mg2+ are used.
The methods making use of the polyanion precipitant have the following common feature.
Specifically, any of the precipitants described is added to serum and is left for 5 to 20 minutes (the time varies according to a precipitant used) so that a sufficient amount of precipitate is generated, and then centrifugal separation is performed for 10 to 15 minutes at a rotational speed of 3000 rpm. After the centrifugal separation, the supernatant liquid (HDL fraction) is extracted, and quantification is carried out at 37° C. by cholesterol chromogenic reaction or the enzyme electrode method.